MicroRNAs (miRNAs) are an abundant class of small RNAs that repress the expression of target messenger RNAs (mRNAs). MiRNAs regulate a variety of biological processes, are essential for mammalian development and have been implicated in numerous diseases. Despite the fact that miRNAs regulate up to 50% of the mammalian genome, the exact mechanism and components of the miRNA-silencing pathway are still poorly understood. To exert their regulatory function, miRNAs are incorporated into a ribonucleoprotein complex termed miRNA- induced silencing complex (miRISC) that is guided to mRNA targets. The effects of miRNA- induced silencing are diverse;while some targets undergo degradation others are translationally repressed. It has become increasingly evident that additional protein cofactors can interact with miRISC and modulate the nature and efficacy of miRISC activity. Among these protein cofactors, members of the TRIM-NHL protein family that control development and stem cell fate in various organisms were recently found to modulate miRNA function. We have identified Trim71, a developmentally regulated member of the TRIM-NHL family, as a novel miRISC- interacting protein that enhances the function of miRNAs and is critical for rapid proliferation of mouse embryonic stem (ES) cells. Our goal is to gain mechanistic insight into the role of Trim71 in miRNA-mediated gene repression and cell proliferation in mouse ES cells. Our preliminary data indicates that Trim71 is a component of a large multi-subunit ribonucleoprotein complex in ES cells. We will employ a variety of approaches to identify the protein and RNA components of Trim71-containing complexes. In addition, we will perform a detailed biochemical analysis of Trim71 to functionally define important protein domains required for interaction with miRISC, enhancement of miRNA function and control of ES cell proliferation. Accomplishing the goals of this proposal will provide a better understanding of how stem cells are regulated at the molecular level and could aid in the development of novel therapeutics. PUBLIC HEALTH RELEVANCE: Stem cells hold great promise for the development of new approaches to combat disease. However, the molecular basis for stem cell self-renewal and differentiation are currently poorly understood. The proposed work will provide novel insight into post-transcriptional regulation of gene expression in embryonic stem cells, and may lead to new therapies to manipulate microRNAs. These studies are relevant to the treatment of cancer, diabetes, developmental disorders and numerous degenerative diseases.